EP2339951A1 - Dispositif, procédé et kit pour la détection in vivo d'un biomarqueur - Google Patents

Dispositif, procédé et kit pour la détection in vivo d'un biomarqueur

Info

Publication number
EP2339951A1
EP2339951A1 EP09787469A EP09787469A EP2339951A1 EP 2339951 A1 EP2339951 A1 EP 2339951A1 EP 09787469 A EP09787469 A EP 09787469A EP 09787469 A EP09787469 A EP 09787469A EP 2339951 A1 EP2339951 A1 EP 2339951A1
Authority
EP
European Patent Office
Prior art keywords
nhs
peg
spacer
biomarker
binding agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09787469A
Other languages
German (de)
English (en)
Inventor
Elisha Rabinovitz
Abraham Rubinstein
Yechezkel Barenholz
Elena Khazanov
Abdel Kareem Azab
Noam Emmanuel
Eylon Yavin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Given Imaging Ltd
Yissum Research Development Co of Hebrew University of Jerusalem
Original Assignee
Given Imaging Ltd
Yissum Research Development Co of Hebrew University of Jerusalem
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Given Imaging Ltd, Yissum Research Development Co of Hebrew University of Jerusalem filed Critical Given Imaging Ltd
Publication of EP2339951A1 publication Critical patent/EP2339951A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
    • A61B1/04—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor combined with photographic or television appliances
    • A61B1/041—Capsule endoscopes for imaging
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
    • A61B1/04—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor combined with photographic or television appliances
    • A61B1/043—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor combined with photographic or television appliances for fluorescence imaging
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0082—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes
    • A61B5/0084—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes for introduction into the body, e.g. by catheters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
    • A61B5/6846—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive
    • A61B5/6847—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive mounted on an invasive device
    • A61B5/6861—Capsules, e.g. for swallowing or implanting

Definitions

  • the invention relates to a device and a system for in-vivo detection of a biomarker in the gastrointestinal tract.
  • the invention further relates to a method for the in-vivo detection of a biomarker in the gastrointestinal tract such as e.g., the ⁇ l-antitrypsin precursor (AlAT biomarker), by using the recognition factor, e.g., trypsin immobilized to a solid surface.
  • the invention further relates to a kit for the in-vivo detection of a biomarker in the gastrointestinal system. /
  • biomarkers expressed in the gastric juice are a -sign of gastric cancer.
  • One example of such a biomarker is human ⁇ l-antitrypsin precursor (AlAT), a 52 Kd member of the serine protease inhibitors (serpins family).
  • AlAT human ⁇ l-antitrypsin precursor
  • serpins family a 52 Kd member of the serine protease inhibitors
  • AlAT is a broad- spectrum protease inhibitor for many serine proteinases, including trypsin. Its proteolytic activity involves cleavage between Met 338 and Ser 359 , which induces a conformational change of AlAT, locking the enzyme and its substrate into a stable, inactive 1:1 enzyme-inhibitor complex.
  • the gastric juice of patients having early and advanced gastric cancer has been found to contain high levels of AlAT.
  • Kits for vitro testing of body fluid samples for the presence of a suspected substance are known in the art. For example, in some cases, diseases, such as cancer, are detected by analyzing blood samples for tumor specific markers, typically, proteins and nucleic acids.
  • a drawback of this method is that the appearance of biomarkers in the blood stream usually occurs at a late stage of the disease, such that early detection is not possible using this method. Moreover, many of the biomarkers are common to several diseases and organs, and therefore their detection in the blood does not allow specific disease detection. Local detection of biomarkers in-vivo in the relevant organ might overcome such drawbacks and enable a sensitive and specific disease detection at early stages.
  • gastric cancer diagnosis requires invasive procedures that include upper GI endoscopy during which biopsies are taken and sent to pathology lab. Such procedures are invasive, time consuming and may miss-detect gastric cancer due to improper sampling. Thus, there is a need in the art for a non-invasive method for detecting gastric cancer at an early stage.
  • WO 09/057120 describes a capsule that can sample intestinal fluids while traversing the gastrointestinal (GI) tract and may perform analysis of the sample for the presence of suspected substances onboard the capsule. Surpassingly, trying to immobilize the recognition factor directly onto the capsule surface, according to some of the embodiments described in WO 09/057120, two significant drawbacks were discovered. The first one is poor surface density (number of recognition factors per unit surface) that can be achieved by techniques common in the art. This drawback may limit the sensitivity that can be achieved by the capsule. Another drawback refers to high non specific adsorption to the surface that may cause poor signal to noise ratio.
  • One of the purposes of this invention is to overcome the above drawbacks.
  • the invention is a device for in-vivo detection of a cancer biomarker in the gastrointestinal system, the device comprising a housing.
  • the housing may comprise an optical window and may enclose a detector or an imager and a light source.
  • an external surface of the optical window may be coated with a polymer.
  • the polymer may have a recognition factor immobilized thereon via a spacer.
  • the imager may be configured to detect changes occurring at the optical window.
  • the invention is a device for in-vivo detection of a biomarker in the gastrointestinal system, the device comprising a housing.
  • the housing may comprise an optical window, a detector or an imager, a light source and a transparent slide.
  • the transparent slide may be made of glass, silica, quartz, cellulose or any transparent plastic or polymer comprising a recognition factor immobilized onto the slide via a spacer.
  • a detector may be configured to detect optical changes on its surface.
  • an imager may be configured to image the transparent slide.
  • the invention is further directed to a system for in-vivo detection of a biomarker in the gastrointestinal system, the system comprising a device according to any of the embodiments detailed above and a transmitter to transmit images from the imager.
  • the system may further comprise a receiving system to receive transmitted signals, and a display to display indication of the presence of a marker in-vivo.
  • the invention includes also a method for the in-vivo detection of the presence of a specific cancer biomarker in the gastrointestinal system of a subject comprising the step of orally administering a device, according to any one of the embodiments detailed above, to the subject.
  • the method may further comprise the step of contacting the orally administered device with a detectable labeled binding agent that binds specifically to the biomarker or contacting the orally administered device with a first binding agent that binds specifically to the biomarker and a second detectable labeled binding agent that binds specifically to the first binding agent.
  • the presence of a bound label as detected by the imager may be indicative to the presence of said specific biomarker in the gastrointestinal system of the subject.
  • the invention further includes a diagnostic kit comprising a device according to any one of the embodiments detailed above, and a binding agent capable of specifically binding a biomarker which is labeled by a detectable label or a combination of a first binding agent that binds specifically to the biomarker and a second detectable labeled binding agent that binds specifically to the first binding agent.
  • the binding agent or the combination may be either contained in a separate container or may be included in the device.
  • the invention is further directed to a transparent film coated by a recognition platform that binds to a specific biomarker.
  • the recognition platform may comprise a polymer and a recognition factor conjugated by a spacer.
  • the invention is further directed to a device for in-vivo detection of a biomarker in the gastrointestinal system, the device comprising a housing, said housing may comprise an optical window and may enclose a light receptor and a light source.
  • an external surface of the optical window may be coated by a polymer.
  • the polymer may have a recognition factor immobilized thereon via a spacer.
  • the light receptor may be configured to detect light changes on the illuminated surface of the optical window.
  • a device for in-vivo detection of a biomarker in the gastrointestinal system is provided.
  • the device may comprise a housing.
  • the housing may comprise an optical window, a light receptor, a light source, and a glass slide.
  • the glass slide may comprise a recognition factor immobilized onto the glass slide via a spacer.
  • the light receptor may be configured to detect light changes on the illuminated surface of the glass slide.
  • the invention is further directed to a system for in-vivo detection, the system comprising a device according to any one of the embodiments detailed above.
  • the system may comprise a transmitter to transmit data from the light receptor.
  • the system may further comprise a receiving system to receive transmitted signals, and a display to display indication of the presence of a marker in-vivo.
  • the invention includes a method for the in-vivo detection of the presence of a specific cancer biomarker in the gastrointestinal system of a subject.
  • the method may comprise the step of orally administering a device according to any one of the embodiments detailed above to the subject.
  • the method may further comprise contacting the orally administered device with a detectable labeled binding agent that binds specifically to the biomarker or contacting the orally administered device with a first binding agent that binds specifically to the biomarker and a second detectable labeled binding agent that binds specifically to the first binding agent.
  • the presence of a bound label as detected by the light receptor may be indicative to the presence of the specific biomarker in the gastrointestinal system of the subject.
  • the invention includes a diagnostic kit comprising a device according to any one of the embodiments detailed above, and a binding agent capable of specifically binding a biomarker.
  • the biomarker may be labeled by a detectable label or a combination of a first binding agent that binds specifically to biomarker and a second detectable labeled binding agent that binds specifically to the first binding agent; wherein the binding agent or the combination may be either contained in a separate container or may be enclosed in the device.
  • the invention includes a polycarbonate or a transparent film coated by a recognition platform that binds to a specific cancer biomarker.
  • the recognition platform may comprise a polymer having a recognition factor immobilized thereon via a spacer.
  • the invention further includes a glass slide that binds to a specific cancer biomarker.
  • the glass slide may comprise a recognition factor immobilized thereon via a spacer.
  • a device for in-vivo detection of a biomarker in the gastrointestinal system comprising: a housing comprising an optical window and enclosing a light receptor and a light source for illuminating in-vivo through the optical window; wherein an external surface of the optical window is coated by a polymer, the polymer having a recognition factor immobilized thereon via a spacer, and wherein the light receptor is configured to detect light changes on the illuminated surface of the optical window.
  • the light receptor in an embodiment of the invention, is a photodetector covered by high pass- or a notch filter configured to detect fluorescent changes on the illuminated surface of the optical window.
  • the light source is one or more LED.
  • the light receptor is an imager or a CMOS.
  • a device for in-vivo detection of a biomarker in the gastrointestinal system comprising: a housing comprising an optical window, a light receptor and a light source for illuminating in-vivo and for illuminating the glass slide, and a glass slide comprising a recognition factor immobilized onto the glass slide via a spacer, wherein the light receptor is configured to detect light changes on the illuminated surface of the glass slide.
  • Figure 1 Shows the chemical structures of the spacers: (A) LPEI (a) and LPEI-max (b), (B) O,O'-Bis[2-(N-Succinimidyl-succmylaxnmo)ethyl] polyethylene glycol 3,000
  • Figure 2 Depicts the grafting of spacer A, spacer B or combination of B and C onto the polyHEMA backbone and the attachment of protein (whether antibody or trypsin) to their active ends.
  • Figure 3 Shows recognition of increasing concentrations (1.25- 10 ⁇ g/ml) of AlAT by rabbit polyclonal anti-Al AT IgG (triangles) or trypsin (circles) coats of polystyrene surface (96 well ELISA plate).
  • (B) Shows specificity of AlAT detection by surface immobilized trypsin as analyzed by 30 ⁇ g/ml of rabbit anti AlAT IgG (circles). Similar concentration of polyclonal rabbit anti-rotavirus IgG served as non-specific control (triangles). In both cases the secondary antibodies was HRP conjugated goat anti rabbit IgG.
  • Figure 4 The effect of crosslinking density (expressed in mol% of ethylene glycol dimethacrylate (EGDMA) used for polyHEMA crosslinking) on the film transparency (expressed in optical density) as measured at a wave length of 600 nm, in a dry and hydrated (three different pH values) state. Shown are the mean values of three experiments ⁇ S. D.
  • Figure 5 Binding of Alexa Fluor 647 labeled hydrazine to polyHEMA films grafted with three concentrations (0, 3.5 and 7 mg/ml) of activated (GA) LPEI spacers.
  • Figure 6 Binding of Alexa Fluor 488 labeled goat anti-rabbit polyclonal IgG to polyHEMA films grafted with: (A) LPEI spacers of increasing molecular weights (2.5 kDa - white columns, 25 kDa - black columns and 250 kDa - light grey columns) and LPEI-max, 4OkDa - dark grey columns. Note the minimal antibody adsorption to polyHEMA films without spacers; (B) Two concentrations of NHS- 2K PEG, four concentrations of NHS- 3K PEG-NHS and two concentrations of a mixture of NHS - 3K ⁇ PEG-NHS with NHS- ⁇ 2K.TPEG. Shown are the mean values of three experiments ⁇ S. D.
  • Figure 7 Capturing Alexa 488 labeled OVA by immobilized (via LPEI, 25 kDa of increasing surface densities) polyclonal rabbit anti- OVA IgG on polyHEMA films.
  • Figure 8 AlAT recognition (expressed in O.D. arbitrary units) by trypsin (1 mg/ml), immobilized by 7 mg/ml LPEI 25 KDa (open triangles), or 100mg/ml NHS- 3 K PEG-NHS (filled squares), or a mixture of lOmg/ml of NHS- 3K PEG-NHS + 100mg/ml ofNHS- 2K PEG (filled circles) to the polyHEMA film, as analyzed by ELISA.
  • the recognition antibody was rabbit anti-AlAT.
  • the secondary antibody was HRP-conjugated anti-rabbit IgG.
  • Figure 9 The reduction in polyHEMA films transparency (expressed in arbitrary O.D. units at 600 nm).
  • A after its grafting with LPEI 25 kDa (light grey columns), activating the spacer with GA (dotted columns), linking IgG to the activated end of the spacer (white columns), or linking trypsin to the activated end of the spacer (dark grey columns);
  • B after its grafting with NHS- 3K PEG-NHS (light grey columns), linking IgG to the activated end of the spacer (white columns).
  • Figure 10 Swelling kinetics of polyHEMA films, crosslinked with increasing amounts of EGDMA, as measured by fluid uptake at pH 1.5 (A) and pH 7.5 (B).
  • Figure 12 The effect of spacer arm density on the glass slide on Alexa Fluor 555 labeled OVA binding.
  • Figure 14 The effect of the NHS-5kPEG spacer on the non-specific recognition, in vitro, of the AlAT by secondary, Alexa Fluor647 conjugated anti-rabbit IgG.
  • Figure 15 Recognizing AlAT in gastric juice by trypsin (lOO ⁇ g/ml) immobilized to SMSA glass slide with a spacers mixture (10 mM NHS-SkPEG-NHS + 50 mM NHS- 2kPEG) or single spacer (NHS-5kPEG, 30 mM). Detection was performed by Alexa Fluor647 conjugated anti-rabbit IgG, at 635 nm (ex), 660 (em). Shown are the mean values of two experiments.
  • Figure 16 The effect of MAL-5KPEG-NHS spacer density (3-3OmM) on the binding of the Alexa Fluor 555 labeled OVA (grey columns) or OVA-SH (black columns) to the SMSA glass slide.
  • Figure 17 The difference in AlAT binding by SH-modified trypsin (1 OO ⁇ g/ml) attached to SMSA glass slide via MAL- 5K PEG-NHS or NHS- 5K PEG spacers, as analyzed by Alexa Fluor 647 conjugated anti-rabbit IgG in SGF. Shown are the mean values of two experiments
  • Figure 18 Binding of Alexa Fluor D68 labeled IgG (two concentrations) to the surface of SMSA-spacer glass slide, previously modified by 30 mM of two types of spacers: NHS- carbonate-NHS or NHS- 3K PEG-NHS. Shown are the mean values of two experiments.
  • Figure 19 The effect of spacer (NHS- 3K PEG-NHS) density on the binding of the Alexa Fluor 568 labeled IgG (25 ⁇ g/ml) to the SMSA glass slide.
  • Figure 20 SDS-PAGE gel electrophoresis of the non-reduced and reduced TRITC- conjugated (Fab) 2 .
  • Figure 21 The binding of increasing concentrations of reduced (Fab:DTT- 1:200, final concentration: 0.5mM) F(ab) 2 fragments and intact (Fab) 2 to the SMSA glass slide pre-treated with 2OmM of MAL-5KPEG-NHS .
  • Figure 22 Is a schematic illustration of an in-vivo detecting device used according to one embodiment of the invention
  • Some embodiments of the present invention are directed to a typically swallowable in- vivo device, e.g., a capsule endoscope.
  • Devices according to embodiments of the present invention may be similar to embodiments described in United States Patent Number 7,009,634, entitled “Device And System For In-vivo Imaging", filed on 8 March, 2001, and/or in United States Patent Number 5,604,531 to Iddan et al., entitled “In-vivo Video Camera System”, and/or in International Application number WO 02/054932 entitled “System and Method for Wide Field Imaging of Body Lumens” published on July 18, 2002, all of which are hereby incorporated by reference.
  • an external receiving unit and processor such as in a work station, such as those described in the above publications could be suitable for use with embodiments of the present invention.
  • Devices and systems as described herein may have other configurations and/or other sets of components.
  • the present invention may be practiced using an endoscope, laparoscope, needle, stent, catheter, etc.
  • Fig.22 schematically illustrates a system according to an embodiment of the invention.
  • the system may include a device 140 having a sensor, e.g., light detector or imager 143 equipped with optical filters to match one or more illumination sources 142 to provide fluorescence detection, a power source 145, and a transmitter 141.
  • device 140 may be implemented using a swallowable capsule, but other sorts of devices or suitable implementations may be used.
  • an external receiver/recorder 112 that include an antenna, a processor, and a display.
  • Transmitter 141 may operate using radio waves; but in some embodiments, such as those where device 140 is or is included within an endoscope, transmitter 141 may transmit/receive data via, for example, wire, optical fiber and/or other suitable methods. Other known wireless methods of transmission may be used.
  • Embodiments of device 140 are typically autonomous, and are typically self- contained.
  • device 140 may be a capsule or other unit where all the components are substantially contained within a housing or shell, and where device 140 does not require any external wires or cables to, for example, receive power or transmit information, hi some embodiments, device 140 may be autonomous and non-remote-controllable; in another embodiment, device 140 may be partially or entirely remote-controllable.
  • device 140 may include in addition to sensor 143 an in-vivo video camera, for example, imager 146 together with optical system 150, which may capture and transmit images of, for example, the gastrointestinal (GI) tract while device 140 passes through the GI lumen.
  • GI gastrointestinal
  • An external receiver/recorder 112 including, or operatively associated with, for example, one or more antennas, or an antenna array, storage unit 119, a processor 114, and a monitor 118.
  • processor 114, storage unit 119 and/or monitor 118 may be implemented in workstation 117.
  • Other lumens and/or body cavities may be imaged and/or sensed by device 140.
  • detector 143 may include, for example, light detector with suitable optical filter adjusted for fluorescence, a Charge Coupled Device (CCD) imager, a Complementary Metal Oxide Semiconductor (CMOS) imager, or other suitable light or image acquisition components.
  • CCD Charge Coupled Device
  • CMOS Complementary Metal Oxide Semiconductor
  • transmitter 141 may transmit/receive via antenna 148.
  • Transmitter 141 and/or another unit in device 140 e.g., a controller or processor 147, may include control capability, for example, one or more control modules, processing module, circuitry and/or functionality for controlling device 140, for controlling the operational mode or settings of device 140, and/or for performing control operations or processing operations within device 140.
  • Power source 145 may include one or more batteries or power cells.
  • power source 145 may include silver oxide batteries, lithium batteries, other suitable electrochemical cells having a high energy density, or the like. Other suitable power sources may be used.
  • power source 145 may receive power or energy from an external power source (e.g., an electromagnetic field generator), which may be used to transmit power or energy to in-vivo device 140.
  • an external power source e.g., an electromagnetic field generator
  • power source 145 may be internal to device 140, and/or may not require coupling to an external power source, e.g., to receive power. Power source 145 may provide power to one or more components of device 140 continuously, substantially continuously, or in a non-discrete manner or timing, or in a periodic manner, an intermittent manner, or an otherwise non-continuous manner. In some embodiments, power source 145 may provide power to one or more components of device 140, for example, not necessarily upon-demand, or not necessarily upon a triggering event or an external activation.
  • transmitter 141 may include a processing unit or processor or controller, for example, to process signals and/or data generated either by imager 146 or sensor 143 or both.
  • the processing unit may be implemented using a separate component within device 140, e.g., controller or processor 147, or may be implemented as an integral part of imager 146, transmitter 141, or another component, or may not be needed.
  • the processing is preformed at the receiver and display unit 112 by an appropriate Digital Signal Processor (DSP),
  • DSP Digital Signal Processor
  • the processing unit may include, for example, a Central Processing Unit (CPU), a microprocessor, a controller, a chip, a microchip, a controller, circuitry, an Integrated Circuit (IC), an Application-Specific Integrated Circuit (ASIC), or any other suitable multi-purpose or specific processor, controller, circuitiy or circuit.
  • the processing unit or controller may be embedded in or integrated with transmitter 141, and may be implemented, for example, using an ASIC.
  • device 140 may include one or more illumination sources 142, for example one Light Emitting Diode (LED) matching the excitation wavelength of the tagged material and another LED or more, "white LEDs", or other suitable light sources to illuminate a body lumen or cavity being imaged.
  • An optional optical system 150 including, for example, one or more optical elements, such as one or more lenses or composite lens assemblies, one or more suitable optical filters, or any other suitable optical elements, may optionally be included in device 140 and may aid in focusing reflected light onto imager 146, focusing illuminated light, and/or performing other light processing operations.
  • illumination source(s) 142 may illuminate in a pre-defined sequence for example in a periodic manner or an otherwise non-continuous manner to enable both: measuring fluorescence signal by sensor 143 and capturing images by imager 146.
  • information sensed by sensor 143 at a certain time period may be displayed on monitor 118 along with the corresponding image information sensed by imager 146 at the same time.
  • information regarding presence of a specific biomarker in the gastrointestinal system, or information regarding presence of a plurality of different biomarkers that is captured by sensor 143 may be displayed on monitor 118 along side an image of the gastrointestinal system.
  • the image information captured by imager 146 may be captured at the same time that the data sensed by sensor 143 was captured.
  • information captured by sensor 143 may be displayed onto the corresponding image captured by imager 146 at the same time that sensor 143 sensed the information.
  • an image of the gastrointestinal system may also show information regarding the presence or lack of presence of a biomarker in-vivo.
  • an image of an area of the GI tract along with the indication of presence of a biomarker may also provide indication on the in-vivo location of the biomarker.
  • the components of device 140 may be enclosed within a housing or shell, e.g., capsule-shaped, oval, or having other suitable shapes.
  • the housing or shell may be substantially transparent or semi-transparent, and/or may include one or more portions, windows or domes which may be substantially transparent or semi-transparent.
  • one or more illumination source(s) 142 within device 140 may illuminate the detection optical window and the excited light detected by sensor 143 while other illumination sources are designed to illuminate a body lumen through a transparent or semi-transparent window or dome that does not contain immobilized recognition factors; and light reflected from the body lumen may enter the device 140, for example, through the same transparent window or dome, or, optionally, through another transparent or semi-transparent portion, window or dome, and may be received by optical system 150 and/or imager 146.
  • Data processor 114 may analyze the data received via external receiver/recorder 112 from device 140, and may be in communication with storage unit 119, e.g., transferring frame data to and from storage unit 119. Data processor 114 may provide the analyzed data to monitor 118, where a user (e.g., a physician) may view or otherwise use the data. In some embodiments, data processor 114 may be configured for real time processing and/or for post processing to be performed and/or viewed at a later time.
  • control capability e.g., delay, timing, etc
  • a suitable external device such as, for example, data processor 114 or external receiver/recorder 112 having a transmitter or transceiver
  • information captured by sensor 143 may be presented with the corresponding image captured by imager 146 at the same time.
  • Monitor 118 may include, for example, one or more screens, monitors, or suitable display units. Monitor 118, for example, may display in addition to the information captured from sensor 143 and/or transmitted by device 140 also one or more images or a stream of images, e.g., images of the GI tract or of other imaged body lumen or cavity.
  • monitor 118 may display, for example, location or position data (e.g., data describing or indicating the location or the relative location of device 140), orientation data, and various other suitable data.
  • location or position data e.g., data describing or indicating the location or the relative location of device 140
  • orientation data e.g., orientation data
  • various other suitable data e.g., orientation data, and various other suitable data.
  • Other systems and methods of storing and/or displaying collected image data and/or other data may be used.
  • device 140 may include few sensors 143 each one configured to detect presence of a different type of biomarker.
  • imager 146 other sensor may be used to, for example, sense, detect, determine and/or measure one or more values of properties or characteristics of the surrounding of device 140.
  • imager 146 may be replaced by a pH sensor, a temperature sensor, an impedance sensor, a pressure sensor, or any other known suitable in-vivo sensor.
  • the in-vivo sensing device is a capsule endoscope.
  • the capsule endoscope typically has a dome shaped optical window at one or both ends of the capsule. Other windows are possible, for example the optical window may be along a side of the device or surrounding the device. Behind the optical window, enclosed within the capsule housing are positioned an image sensor or another light receptor, an optical system for focusing images onto the image sensor and at least one illumination source for illuminating the gastrointestinal (GI) tract through which the capsule endoscope is propagating.
  • GI gastrointestinal
  • the device may be any capsule.
  • a device for in-vivo detection of a biomarker in the gastrointestinal tract comprises a housing.
  • the housing may comprise an optical window, and behind the optical window, enclosed within the housing may be positioned a light receptor e.g. imager.
  • an external surface of the optical window may be coated by a transparent or preferably semitransparent polymer.
  • the polymer may have a recognition factor immobilized thereon via a spacer.
  • the imager may be configured to image the optical window.
  • the external surface of the optical window is coated.
  • the optical window is made of a plastic such as Isoplast® or polycarbonate.
  • Other solid phase substrates may be used, for example, glass, silica, or other biocompatible plastics, such as polypropylene and polystyrene.
  • the recognition factor is attached to a recognition platform positioned across the illumination source and light detector/receptor; such platform may be made of various materials, organic or inorganic or a combination of both.
  • the recognition platform may be included in the device by any appropriate manner, such as a coating, enclosing in a compartment, etc.
  • Suitable materials include, but are not limited to, glasses, ceramics, plastics, metals, alloys, carbon, papers, agarose, silica, quartz, cellulose, polyacrylamide, polyamide, and gelatin, as well as other polymer supports, other solid-material supports, or flexible membrane supports.
  • Polymers that may be used as a substrate include, but are not limited to: polystyrene; poly(tetra)fluoroethylene (PTFE); polyvinylidenedifluoride; polycarbonate; polymethylmethacrylate; polyvinylethylene; polyethyleneimine; polyoxymethylene (POM); polyvinylphenol; polylactides; polymethacrylimide (PMI); polyalkenesulfone (PAS); polypropylene; polyethylene; polydimethylsiloxane; polyacrylamide; polyimide; and various block co-polymers.
  • the substrate or support can also comprise a combination of materials, whether water-permeable or not, in multi-layer configurations.
  • the polymer is polyHEMA.
  • the recognition platform is a polyHEMA film crosslinked with an appropriate amount of ethylene glycol dimethacrylate (EGDMA), e.g., 5 mol%.
  • EGDMA ethylene glycol dimethacrylate
  • a device and a system for in-vivo detection of a cancer biomarker in the gastrointestinal system comprising: a housing comprising an optical window made of glass slide comprising a recognition factor immobilized onto the glass slide via a spacer, behind the optical window, enclosed within the capsule housing are positioned light receptor e.g. imager wherein the imager is configured to image the glass slide.
  • the glass slide according to the embodiment of the invention may be Super MaskTM SuperAmine 2 (SMSA), SuperMaskTM SuperAmine, SuperMaskTM SuperClean 2, SuperMaskTM 16 SuperAldehyde 2, SuperMaskTM 16 SuperEpoxy 2 each containing 4, 12, 16, 24, 48, 64, or 192 hydrophobic wells.
  • the Super MaskTM SuperAmine 2 (SMSA) glass slide is characterized by ultra-low intrinsic fluorescence and background noise, containing high density (2 x 10 13 ) of charged amino groups/mm 2 .
  • the device includes a chamber in which the glass slide such as SMSA is found, e.g., the glass slide is positioned behind a capsule's dome-shaped optical window, which has the ability to allow gastric fluids flow there through.
  • the free flow of gastric fluids through the capsule typically allows the biological recognition reaction to take place on top of shielded glass surface (i.e. the glass slide).
  • a capsule may comprise a dark background which may reduce or eliminate the interference of tissue auto-fluorescence with the fluorescence emitted by the presence of the biomarker; thus increasing signal to noise ratio.
  • the system in some embodiments of the invention includes the device enclosing the glass slide according to the embodiments of the invention and a transmitter to transmit images from the imager; a receiving system to receive transmitted signals; and a display to display indication of the presence of a marker in-vivo.
  • the biomarker is, for example, without limitation, ⁇ l -antitrypsin precursor (AlAT), carcinoembryonic antien (CEA) or CA 19-9.
  • AlAT ⁇ l -antitrypsin precursor
  • CEA carcinoembryonic antien
  • CA 19-9 a biomarker to gastric cancer or to any other disease that is known or will be known in the art.
  • the spacer molecules used for immobilizing the recognition factor specific to the biomarker such as a protein, polypeptide, or antibody on the polyHEMA film include (a) linear poly(ethyleneimine) (LPEI); (b) O 5 O'- bis[2-(N-succinimidyl-succinylamino)ethyl]polyethylene glycol (NHS- 3K PEG-NHS); (c) a combination of NHS- 3K PEG-NHS and O-[(N-succinimidyl)succinyl-aminoethyl]-O'- methylpolyethylene glycol (NHS- 2K PEG); (d) Amino-PEG-Carboxylic acid; (e) Boc- piOtected-Amino-PEG-Carbonate-NHS; (f) Maleimide-PEG-Carbonate-NHS; (J) Hydroxy- PEG- Aldehyde; (LPEI); (b) O 5 O'-
  • the recognition factor may be a protein, a polypeptide, a polynucleotide or anti-biomarker antibody, or a substrate, that specifically bind to the biomarker.
  • binding refers to the interaction between a protein, polypeptide, peptide or carbohydrate and a binding molecule, such as a ligand, a substrate, an antibody, a peptide or an aptamer.
  • a binding molecule such as a ligand, a substrate, an antibody, a peptide or an aptamer. The interaction is dependent upon the presence of a particular structure (i.e., an antigenic determinant or epitope or substrate binding site in case of an enzyme) of the protein that is recognized by the binding molecule.
  • Methods of coupling the proteins, polypeptides or anti-biomarker antibodies to the reactive end groups on the surface of the substrate or on the spacer include reactions that form linkage such as thioether bonds, disulfide bonds, amide bonds, carbamate bonds, urea linkages, ester bonds, carbonate bonds, ether bonds, hydrazone linkages, Schiff-base linkages, and noncovalent linkages mediated by, for example, ionic or hydrophobic interactions.
  • the form of reaction will depend, of course, upon the available reactive groups on both the recognition factor/spacer and the antibodies.
  • the trypsin recognition factor may be immobilized onto the recognition platform's surface using any appropriate spacer molecule for capturing the AlAT.
  • the trypsin is anchored on a transparent polyHEMA film, aimed at detecting AlAT on the capsule surface by the use of spacers which are O-[(N-succinimidyl)succinyl-aminoethyl]-O'-methylpolyethylene glycol (NHS- 2K PEG) or O,O'-bis[2-(N-succinimidyl-succinylamino)ethyl]polyethylene glycol (NHS- 3K PEG-NHS).
  • the specific interaction between the immobilized trypsin and AlAT is followed by the addition of primary and labeled or fluorescent secondary antibodies in order to generate a detectable signal.
  • the invention may be also conducted by the use of a labeled primary antibody.
  • the recognition factor attaches to the biomarker found in the gastric fluid, it is necessary to quantify the amount of the biomarker recognized by the recognition factor. This may be done by any appropriate method known in the art.
  • a method for the in-vivo detection of the presence of a biomarker in the gastrointestinal tract of a subject comprising the steps of: orally administering a device according embodiment provided herein; contacting the orally administered device with a detectable labeled binding agent (e.g. primary antibody) that binds specifically to the biomarker or contacting the orally administered device with a first binding agent (e.g. first antibody) that binds specifically to the biomarker and a second detectable labeled binding agent (e.g. secondary antibody) that binds specifically to the first binding agent; wherein the presence of a bound label as detected by the imager is indicative to the presence of the specific biomarker in the gastrointestinal tract of the subject.
  • a detectable labeled binding agent e.g. primary antibody
  • a first binding agent e.g. first antibody
  • a second detectable labeled binding agent e.g. secondary antibody
  • the label for the detection of the binding may be a radioisotope, a fluorescent agent, a magnetic bead, gold particles as well as other metal colloidal particles or other appropriate detectable agent or an enzyme label.
  • Fluorescent labels include, for example, Fluorescein, FITC , Indocyanine green (ICG), Coumarin (e.g., Hydroxycoumarin, Aminocoumarin, Methoxycoumarin), R-Phycoerythrin (PE), Fluorescein, FITC, Fluor X, DTAF, Auramine, Alexa (e.g., Alexa Fluor 350, 430, 488, 532, 546, 555, 568, 594, 633, 647, 660, 680, 700, 750), BODIPY-FL, Sulforhodamine, Carbocyanine (e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7), Rhodamine, XRITC, TRITC, Li
  • Isotope labels include 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 18 Re.
  • Enzyme labels include peroxidase, beta-glucuronidase, beta-D-glucosidase, beta-D- galactosidase, urease, glucose oxidase plus peroxidase, and alkaline phosphatase. Enzymes can be conjugated by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde, and the like.
  • Enzyme labels can be detected visually, or measured by calorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques.
  • Other labeling systems such as avidin/biotin, colloidal gold (e.g., NANOGOLD), Tyramide Signal Amplification (TSA), are known in the art, and are commercially available (see, e.g., ABC kit, Vector Laboratories, Inc., Burlingame, Calif; NEN Life Science Products, Inc., Boston, Mass.; Nanoprobes, Inc., 95 Horse Block Road, Yaphank, N. Y.). The use of any of those labels is subjected to toxicology tests and the approval of the health authorities.
  • the measurement of the fluorescent signal may be performed by any appropriate means, including a miniature spectrophotometer or a photomultiplier or a narrow band illumination source and a photodetector covered by high pass- or a notch filter blocking the excitation light and detecting only the emission light capable of recording the fluorescent signal produced by the reaction between the captured biomarker and the fluorescent preliminary or secondary antibody.
  • a miniature spectrophotometer or a photomultiplier or a narrow band illumination source and a photodetector covered by high pass- or a notch filter blocking the excitation light and detecting only the emission light capable of recording the fluorescent signal produced by the reaction between the captured biomarker and the fluorescent preliminary or secondary antibody.
  • the colored binding agent will be in the field of view of the image sensor and may appear as a colored spot or other shaped mark in an image being obtained by the image sensor also termed here as "imager".
  • the presence of the labeled binding agent which is typically an antibody may be detected, either by being viewed and imaged by the image sensor of the capsule endoscope or by other suitable detecting means which may be included in the capsule endoscope, for example, other optical detectors or a radiation detector.
  • Data sensed by the in-vivo device are transmitted in an embodiment of the invention to an external receiver and are viewed and/or analyzed by a processor outside the body.
  • Data sensed by the device may include indication of the presence of the biomarker.
  • the presence of the binding agent may be indicative of the presence of the marker in the lumen being examined and as such may indicate to a physician that the patient being examined may be in danger of developing cancer or other pathologies.
  • the reaction from the step of exposing the coated device or the device enclosing the glass slide to a primary antibody and measuring the detection lasts no more than 30 minutes. According to an embodiment of this invention, the reaction lasts no more than 20 minutes. According to an embodiment of this invention, the reaction lasts no more than 15 minutes.
  • fiuorescently labeled polystyrene beads may be used in order to detect fluorescent signals from wells in the SMSA glass slide, onto which FluoSpheres® beads were attached specifically.
  • FluoSpheres® beads containing surface- pendent carboxylic moieties, making them suitable for covalent coupling of proteins and other amine-containing biomolecules
  • Other types of "nanocontainers” e.g. iron oxide, gold particles and polysaccharide based Nanoparticles.
  • a kit comprising: a device according to the embodiment of the invention and a binding agent capable of specifically binding a biomarker which is labeled by a detectable label or a combination of a first binding agent that binds specifically to a biomarker and a second detectable labeled binding agent that binds specifically to the first binding agent; wherein the binding agent or the combination are either contained in a separate containers or are included in the device, hi an embodiment of the invention, if the binding agent or the combination of a first and a second binding agent are in separate containers, a leaflet is attached explaining the instructions for oral administration thereof.
  • the binding agent may be in any other suitable form, such as in a powder, spray or suspension or in a tablet.
  • a transparent film coated by a recognition platform that binds to the specific biomarker wherein the recognition platform comprises a polymer and a recognition factor conjugated by a spacer.
  • the transparent film may be coating any capsule.
  • a polycarbonate film coated by a recognition platform that binds to the specific biomarker wherein the recognition platform comprises a polymer and a recognition factor conjugated by a spacer.
  • the polycarbonate film may be coating any capsule.
  • a glass slide that binds to a specific cancer biomarker; wherein the glass slide comprises a recognition factor immobilized thereon via a spacer.
  • the glass slide may be enclosed in any capsule.
  • the in-vivo device may include a sensor such as a sensor of electrical charge to sense a change in electrical charge which may indicate a change in the configuration of the recognition factor due to its interaction with the biomarker.
  • Administering a device in-vivo may be done in any suitable way such as by swallowing (by the patient) or otherwise inserting the device into the patient's GI tract by attaching it to an endoscope or any other suitable in-vivo device.
  • the timing of the different administrations may be planned such to allow sufficient time for the recognition factor to bind the biomarker and only then for the biomarker- recognition factor complex to bind the tagged binding agent.
  • the invention also relates in one of its embodiment to a device, system and method for detection the biomarker, in which two or more different recognition factors are attached to the recognition platform.
  • the detection of two biomarkers more is possible by administering one device.
  • the recognition factors may be attached to the coating onto the optical window or elsewhere in the capsule or to a solid support enclosed in the device or to any combination thereof.
  • the labeled primary antibody or the secondary antibody may be detected by one or more of the methods provided herein.
  • an acid reducing agent may be administered to the patient.
  • Acid reducing agents such as known antacids (e.g., Maalox, Rolaids etc.) will typically raise and buffer the pH level in the stomach, thus providing a more stable environment for the recognition factor and the binding agents (typically proteins) and for the markers themselves.
  • acid reducing agents may neutralize pepsin in the stomach and, at least in part, may inhibit the activation of protease precursors that are secreted from the pancreas into the bowel, thus providing an environment essentially free of active pepsin for the procedure of the invention.
  • a pH level of between about 6.0 to about 7.4 may be desirable.
  • pH in the range of 6-8 is optimal for stable trypsin (as well as other relevant proteases that can bind A1AT)/A1AT complex formation.
  • other pH levels may also be obtained according to embodiments of the present invention.
  • a pH of above 5.5 may be obtained.
  • FIG. 5 and 6 show that the presence of the spacer arms was important for binding significant amounts of the fluorescent molecule, which do not bind well to a polyHEMA film that did not contain a spacer.
  • Figure 6 further shows that the LPEI spacer was more effective that the NHS-3KPEG-NHS or its mixture with NHS-2KPEG in binding the goat anti-rabbit polyclonal IgG to the polyHEMA films.
  • OVA ovalbumin
  • LPEI linear polyethyleneimine
  • MAX polyethyleneimine "MAX”
  • H+L Alexa-Fluor 488 labeled goat anti-rabbit IgG
  • OVA ovalbumin
  • ELISA 96 wells plates were coated with 10 ⁇ g/ml of rabbit polyclonal anti AlAT IgG (phosphate buffer solution (PBS) pH 7.4, 37 0 C, 45minutes) and other ELISA 96 well were coated with 10 ⁇ g/ml of trypsin (PBS, pH 5, 37 0 C, 45 minutes). After a PBS rinse, each plate was washed twice with PBS supplemented with Tween-20 (0.05%). Non specific binding was blocked with 10% w/v (bovine serum albumin) BSA in PBS (37 °C, 45 minutes).
  • PBS phosphate buffer solution
  • Pillcam capsules (Given Imaging, Yokneam, Israel). The molds were dried at room temperature (24 h) to obtain transparent films, 0.2-0.5 mm thick (measured by Mitutoyo micrometer, Aurora, IL, USA), depending on the concentration of EGDMA used. The larger the concentration of EGDMA, the thinner the firm obtained. A decision on the optimal EGDMA concentration was taken after comparing the physical properties of the three types of films, originating from the three different concentrations of EGDMA tested.
  • EGDMA amount on the modulus of elasticity, adherence to polycarbonate capsule and the stability of the film coat on top of the polycarbonate capsules.
  • Equation 1 As determined gravimetrically (Equation 1), after continuous stirring of coated polycarbonate dome (24 h, 37 0 C) in SGF (no pepsin). Shown are the mean values of 4 separate measurements ⁇ S.D.
  • the LPEI spacers (see Figure 1) (both LPEI and LPEI-max) were attached to the polyHEMA films (see Figure 2) by immersing (gentle shaking, 55 0 C, 16 h), separately, the dry films with increasing concentrations (3.5, 7 or 14 mg/ml) of the various spacers, followed by a water rinse to remove non-reacted residues. Spacer attachment to the polyHEMA films was verified by elemental analysis (Table III), which revealed that, depending on the LPEI type, total nitrogen content was in the range of 0.39-0.58%, corresponding to 4.33-6.44 % of total LPEI bound to the polyHEMA film. Table III: Microanalysis of the LPEI-treated polyHEMA films
  • LPEI spacers required activation by gluteraldehyde (GA) of the grafted molecules, to enable the formation of an imine bond between the spacer and the protein's primary amine (see Figure 2).
  • the end amine groups of the grafted LPEI or the LPEI-Max spacers were activated by bathing (room temperature, 45 minutes) the modified films in 1% w/v GA in water with a subsequent water rinse. Activation was verified by incubating (37 0 C, 2 h) the films (4 mg) with 100 ⁇ L of 5 ⁇ g/mL of Alexa Fluor 647 labeled hydrazine, followed by a water rinse.
  • FIG. 5 shows that the fluorescent hydrazine reacted with the activated polyHEMA films in a spacer-density dependent manner and that the presence of activated LPEI grafts was important for binding significant amounts of the fluorescent molecule (5.5- and 8-fold binding of the films containing 3.5 and 7 mg/ml of LPEI respectively, compared with polyHEMA control film which was treated with GA but did not contain a spacer.
  • Unbound antibody was removed by washing with PBS containing 0.1% w/v Tween-20 and the existence of bound fluorescent IgG was verified spectrofluorimetrically (excitation: 485 nm, emission: 525 nm) in a microplate reader, using GA-treated polyHEMA (no spacer grafts) as controls.
  • polyclonal rabbit anti-OVA IgG was grafted onto the polyHEMA film using increasing concentrations (elevated surface density) of the LPEI 25 kDa spacer.
  • polyclonal rabbit anti-OVA was conjugated to the activated polyHEMA-LPEI film by its incubation (PBS pH 7.4, 4 0 C, overnight) with 15 ⁇ g/ml of the antibody, followed by a Tween-20 (0.1%w/v in PBS) rinse to remove unbound residues and 1% w/v dry milk in PBS (room temperature, 2 h) to block nonspecific protein binding.
  • the PEG-based spacers [00108] In separate studies increasing concentrations (10 -100 mg/ml) of NHS- 3K PEG-NHS, or a mixture of NHS- 3K PEG-NHS with NHS- 2K PEG in PBS (pH 7.4), were immersed (gentle shaking, 3h, 25 0 C) with the dry polyHEMA films followed by a water rinse to remove non- reacted residues. See Figure 1 for the structures of the PEG spacers and Figure 2 for the grafting to the polyHEMA.
  • Unbound antibody was removed by a Tween-20 (0.1%w/v in PBS) rinse.
  • the existence of bound fluorescent IgG was verified spectrofluorimetrically (excitation: 485 nm, emission: 525 nm) in a microplate reader, using NHS- 2K PEG-coated film as control.
  • the HRP conjugated second antibody (1:5000 in PBS) was then added to the wells and the plate was rinsed and reacted with TMB reagent (in citrate buffer, pH 5 to a final volume of 100 ⁇ l/well). The reaction was stopped with H 2 SO 4 IM (100 ⁇ l/well). Color intensity was measured in a microplate reader at 450 ran.
  • Example 6 The SMSA glass slide recognition platform and its spacer arms
  • Fluorescent intensity of the SMSA substrate slides platform was measured by the GenePix Pro 4000 scanner (Axon Instruments, Inc., USA). [00119] The ability of the SMSA- MAL- 5K PEG- NHS products to interact with proteins was examined by incubating the spacer-grafted platforms with 50 ⁇ L of reduced TRITC - conjugated AffiniPure F (ab') 2 fragment of the goat anti-mouse IgG (H+L) (25-100 ⁇ g/mL), dissolved in PBS, pH 6.5 at 37 0 C for one hour. Unbound protein was removed by Tween-20 (0.1%w/v in PBS) rinse. Fluorescent intensity of the SMSA substrate slides platform was measured in Microarray Axon scanner.
  • Example 7 AlAT detection by immobilized trypsin on the glass slide and the selection of a spacer mixture
  • Nonspecific binding was blocked by BSA (1% w/v in PBS for one hour at 37 0 C).
  • Trypsin attachment to the surface of the SMSA glass slides modified with MAL- 5K PEG- NHS (2OmM) was performed by incubation (37 0 C, one hour) with trypsin (100 ⁇ g/ml in PBS, pH 5) bearing sulfhydryl groups. Unbound trypsin was removed by Tween-20 (0.1%w/v in PBS) rinse. Nonspecific binding was blocked by BSA (1% w/v in PBS for one hour at 37 0 C).
  • the amount of recognized A 1 AT was analyzed by sandwich ELISA 5 using rabbit anti- AlAT in SGF.
  • a hetero-bi-functional product was be used instead of the homo-bi-functional NHS-3KPEG-NHS spacer.
  • the specific spacer used is N-[(3-maleimido-l- oxopropyl)aminopropyl- ⁇ -(succinimidyloxy carboxy) polyoxyethylene glycol 5'00O (MAL- 5KPEG-NHS).
  • MAL- 5KPEG-NHS N-[(3-maleimido-l- oxopropyl)aminopropyl- ⁇ -(succinimidyloxy carboxy) polyoxyethylene glycol 5'00O
  • a thiol (-SH) group was introduced into the trypsin molecule.
  • amine groups of trypsin and the proteinecious probe, fluorescently- labeled OVA555 were substituted with a thiol group, by using N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP) and dithiothreitol (DTT).
  • SPDP N-succinimidyl-3-(2- pyridyldithio)propionate
  • DTT dithiothreitol
  • Trypsin modification was performed according to the 2,2'-dithiodipyridine (DTDP) method described above. According to this method 2 SH groups were attached to each trypsin molecule. Elevating this number was found to interfere with the AlAT recognition (as was observed when 7 thiol groups were inserted). [00132] Detection of increasing amounts of AlAT by the new detecting platform was measured in SGF buffered with carbonate buffer solution, assayed by the sandwich ELISA method, employing rabbit anti-AlAT IgG and Alexa Fluor 647 conjugated anti-rabbit IgG.
  • DTDP 2,2'-dithiodipyridine
  • Free DTT was separated from the modified protein with the aid of the Microcon YM- 10 ultrafiltration device by centrifugation for 30 min, 14000 rpm at 2O 0 C. Collected modified protein was resuspended in PBS (pH 6.5) and stored at 4 0 C until used. The integrity of the reduced F(ab') 2 was determined by non-reduced SDS-PAGE (12.5%) using Vertical Gel Electrophoresis System (Bio-Rad). The gel was stained with Coomassie brilliant blue R250. Control experiments were run using unreduced niAb F(ab') 2 .

Abstract

Cette invention concerne un dispositif et un système pour la détection in vivo d'un biomarqueur dans les voies gastro-intestinales. L'invention concerne, en outre, un procédé pour la détection in vivo d'un biomarqueur dans les voies gastro-intestinales tel que, par exemple, le précurseur de l'α1-antitrypsine (biomarqueur A1AT), au moyen du facteur de reconnaissance, par exemple, trypsine immobilisée sur une surface solide. L'invention concerne enfin un kit pour la détection in vivo d'un biomarqueur dans le système gastro-intestinal.
EP09787469A 2008-07-10 2009-07-09 Dispositif, procédé et kit pour la détection in vivo d'un biomarqueur Withdrawn EP2339951A1 (fr)

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